7. Project Summary/Abstract Fasting interventions improve health in mice and in humans. Given that adult stem cells coordinate tissue adaptation, understanding the mechanism(s) that mediate the fasting response has important implications for enhancing tissue repair after injury and in aging where stem cell function declines. In the mammalian intestine, LGR5+ intestinal stem cells (ISCs) drive the rapid renewal of the intestinal lining. We previously showed that fasting augments ISC function by inducing a peroxisome proliferator-activated receptor delta (PPAR?) driven fatty acid oxidation (FAO) program. However, the in vivo role of PPAR? or downstream effector(s) of FAO metabolism that mediate the ISC fasting response remains unknown. In this proposal, we will test the hypotheses that 1) PPAR? signaling is necessary for the in vivo ISC fasting response, 2) PPAR?- activated FAO stimulates intestinal stemness through the production of the ketone body ?-hydroxybutyrate (?OHB) in fasting, and 3) changes in the gut microbiome are necessary, sufficient or both in mediating the ISC fasting response. In support of these notions, we find that PPAR? agonist treatment emulates the effects of fasting on ISCs. Furthermore, we find that enzymes of the ketogenic pathway that produce the ketone metabolite ?OHB, including its rate-limiting step HMGCS2 (3-hydroxy-3-methylglutaryl-CoA synthetase 2), highly enrich for LGR5+ ISCs. Also, elevation of HMGCS2 expression and ?OHB levels upon fasting correlates with enhanced ISC function while loss of HMGCS2 dampens ISC capacity to propagate organoids and biases their differentiation towards the secretory lineage. Importantly, these deficits can be rectified by ?OHB treatment in cultures. Thus, these observations provide a possible pathway for fasting through the modulation of HMGCS2-mediated ketogenesis to augment the regenerative function of ISCs. Lastly, fasting regimens are known to alter the gut microbiome composition but the extent to which these changes underlie the ISC fasting response requires elucidation. Remaining questions regarding the specific in vivo role of PPAR? as an upstream regulator of HMGCS2 expression and ISC fasting response, the in vivo role of HMGCS2 and ?OHB as mediators of intestinal stemness and the contribution of the fasting gut microbiota in these processes.